Systems and methods for encapsulation and multi-step processing of biological samples

ABSTRACT

The present invention relates to methods and systems for isolation of species in semi-permeable capsules and processing of encapsulated species through series of steps and/or reactions. To produce capsules, first aqueous two-phase system (ATPS) droplets are generated using microfluidics system and then the hydrogel shell layer is hardened by inducing polymerization. As exemplified in this invention to achieve concentric ATPS droplet formation density-matched PEGDA and Dextran polymer solutions can be used. Once a capsule is formed, its composition can be changed by adding new reagents or replacing out old ones (e.g. by resuspending capsules in desired aqueous solution). The hydrogel shell of semi-permeable capsules can be dissolved at selected step during multi-step procedures in order to release the encapsulated species. The present invention exemplifies the isolation of individual cells within capsules and using the encapsulated cells for genotypic and phenotypic analysis. Finally, the present invention also exemplifies the use of capsules in multi-step procedures to perform complex biological reactions.

FIELD OF THE INVENTION

The present invention is directed to methods and systems for isolationof species such as cells, bacteria, viruses, nucleic acids, biochemicalcompounds, and/or other materials in semi-permeable capsules, andprocessing the encapsulated species through multi-step procedures toperform sequential reactions. The encapsulated species can be releasedfrom capsules at a desirable step upon treatment with external stimuli.The method revealed here exemplifies the use of capsules for genotypicand phenotypic analysis of individual cells.

BACKGROUND OF THE INVENTION

High-throughput processing and analysis of biological samples at thesingle-cell or single-molecule resolution has important applications inmany branches of life sciences. Compartmentalization of cells, DNA,enzymes or molecules to water-in-oil droplets or other forms ofcompartments enables massively parallel analysis with a throughputorders of magnitude higher when compared to 96-well microtiter plates.However, many molecular biology methods are built on sequential andmulti-step sample processing in order to initiate, modify or terminate areaction. As a result, not all molecular biology workflows can be easilytransferred to droplet or other types of emulsion-based formats.Although some solutions such as droplet fusion, reinjection, andsplitting can enable multi-step procedures (e.g. to add new reagents toa preformed droplet), yet the required expertise and complexity offluidic operations limits the broader use of such approaches. Sequentialsample processing can become very challenging when encapsulated cells,or their genetic material, have to be processed through a series ofindependent reactions. For example, for the amplification and/oranalysis of genetic material of encapsulated cells it may be necessaryto perform cell lysis, a step that might be inhibitory or detrimental tosubsequent enzymatic step(s). As a result, it is advantageous to have amethod and/or system that would enable buffer/reagent exchange and/orremoval of lysis reagents, before nuclei acid analysis/amplificationstep. Considering current state-of-the-art there is unmet need formethods and systems that would enable multi-step processing ofencapsulated entities (e.g. cells). The invention revealed here isrelated to production of capsules with semi-permeable hydrogel shell anduse of the said capsules for processing biological samples, asexemplified by performing genotypic and phenotypic analysis onindividual cells.

Previous attempts to produce capsules suitable for multi-step biologicalreactions have not been successful or practical. Although numerousreports have shown generation of semi-permeable capsules composed ofvariety of polymers, yet to the best of our knowledge no capsules havebeen shown or applied in multi-step reactions to process of, or performanalysis on, encapsulated entities such as cells, biomolecules, etc.

For example, Vijayakumar, K., Gulati, S., Demello, A. J. & Edel, J. B.Rapid cell extraction in aqueous two-phase microdroplet systems. Chem.Sci. 1, 447-452 (2010) applied droplet microfluidic system to generatean aqueous two-phase system (ATPS) in which aqueous droplets consist oftwo phases: PEG-rich and Dextran-rich. They demonstrated T lymphoma cellpartitioning between two layers. However, the authors have not producedsemi-permeable capsules.

Tamminen, M. V. & Virta, M. P. J. Single gene-based distinction ofindividual microbial genomes from a mixed population of microbial cells.Front. Microbiol. 6, 1-10 (2015). The method of generating capsules issignificantly different from the one described in this invention. Inthis work bacteria are encapsulated to acrylamide hydrogel beads, thenthe beads carrying embedded bacteria are re-suspended in warm agaroseand emulsified again that leads to capsules with a hydrogel corecomposed of acrylamide and hydrogel-shell composed of agarose. Theauthors show that the hydrogel core can be dissolved by DTT, as a resultforming liquid core capsules with agarose shell. The method and systemrevealed here involves different steps and generation of capsules relieson microfluidic systems.

Ma, S. et al. Fabrication of microgel particles with complex shape viaselective polymerization of aqueous two-phase systems. Small 8,2356-2360 (2012). Although the authors have used similar polymers,Dextran and PEGDA to produce aqueous two-phase system (ATPS) dropletsthey presented a method for fabricating micro particles with a concaveshape. In one aspect, the invention comprises a method for producingcapsules but the biological samples cannot be contained in such openparticles.

Watanabe, Motohiro, and Ono, Microfluidic Formation of HydrogelMicrocapsules with a Single Aqueous Core by Spontaneous Crosslinking inAqueous Two-Phase System (ATPS) Droplets. Langmuir, 2019. The authorshave demonstrated the fabrication of monodisperse tetra-armpoly(ethylene glycol) (tetra-PEG) hydrogel microcapsules with an aqueouscore and a semi-permeable hydrogel shell through the formation ofaqueous two-phase system (ATPS) droplets consisting of Dextran(DEX)-rich core and tetra-PEG macromonomer-rich shell, followed byspontaneous cross-end coupling reaction of tetra-PEG macromonomers inthe shell. The workflow of capsule generation has similarities with themethods and systems reported here. However, the authors have not shownany of the biological applications such as analysis and processing ofcells or biological samples, or the possibility of using capsules formulti-step reactions.

In the following sections the invention reveals a few, but not limitedto, examples of semi-permeable capsule production, encapsulation ofspecies (such as cells), the use encapsulated-species in multi-stepprocesses and sequential reactions, genotypic and phenotypic analysis ofindividual cells in a massively parallel fashion and other applications.

SUMMARY OF THE INVENTION

The present invention is directed to systems and methods for productionof semi-permeable capsules, the capsule use for encapsulation of cellsand other biological materials, and for high-throughput processing ofencapsulated material in multi-step operations. To form the capsules,first the liquid droplets are generated using microfluidics platform,then liquid-liquid phase separation is allowed to occur in droplets toform so called aqueous two-phase system (ATPS) droplets, then the shellof capsules is hardened by inducing polymerization of one of the phasesof the ATPS droplet. As revealed in this invention, the capsule mayconsist of Dextran-rich solution that forms a capsule's core, andpolyethylene glycol diacrylate (PEGDA) polymer-based shell. However,other combinations and polymers are also possible to use as should beevident from the capsules production steps revealed here. To exemplifythe formation of the shell in ATPS droplets we use photo-illumination,as a chemically neutral measure to induce PEGDA polymerization and formhardened shell. Resulting capsules contain liquid-like core enriched inDextran phase and hydrogel-shell enriched in PEGDA. The core of capsulescan become more viscous after polymerization due to the presence ofresidual PEGDA and/or in some cases may form a hydrogel mesh. Asrevealed here the resulting capsules can be used in multi-step reactionsto process cells and/or various biological materials (e.g. enzymes,proteins, viruses, nucleic acids, etc.). For example, the capsules canbe used to isolate individual cells, to amplify the nucleic acids ofencapsulated cells and perform other reactions required for genotypicanalysis. In yet another example, capsules can be used for growing cellsin capsules to perform phenotypic or genotypic screens and analysis. Asshown here, single-cells can be expanded into micro-colonies. In allthese analyses and operations, the ability to perform multi-stepreactions on many capsules at once is an essential part of thisinvention. When suspended and washed in aqueous buffer multiple timesthe capsules are stiff enough to withstand mechanical stress and remainhighly uniform. As revealed here the semi-permeable capsules can be usedto perform genotypic and phenotypic analysis of individual bacterialcells in a massively parallel fashion. Furthermore, capsules sustainedmultiple temperature cycles during PCR as well as share forces generatedduring flow cytometry. Therefore, the invention is related to the use ofsemi-permeable capsules to isolate cells and/or biological material andsamples for further multi-step processing and/or analysis.

In one aspect, the invention comprises a method for forming/providing afluidic droplet containing the species, causing a separation into innerand outer phases of the fluidic droplet containing the species, inducingthe gelation of the outer phase of the fluidic droplet containing thespecies, and performing multi-step reaction(s) and/or processing on theencapsulated species.

The “species” herein refers to cells, bacteria, viruses, DNA, RNA,proteins, biological material or biochemical compounds that will beprocessed in multi-step reaction.

In one exemplary embodiment, the invention comprises a microfluidicsystem for the production of liquid droplets containing the species.Such system comprises:

-   -   (i) An inlet for continuous phase (carrier oil);    -   (ii) An inlet for the first fluid;    -   (iii) An inlet for the second fluid;    -   (iv) A nozzle or a flow-focusing junction where droplet        formation occurs;    -   (v) A droplet collection outlet.    -   (vi) Channel connecting the nozzle with the collection outlet.    -   (vii) The species are provided with first fluid, with second        fluid or with both.

In another aspect, the invention comprises the method for the formationof liquid droplets containing the species:

-   -   (i) Injection of a first fluid (Phase I solution);    -   (ii) Injection of a second fluid (Phase II solution);    -   (iii) Injection of a carrier oil;    -   (iv) Bringing carrier oil, Phase I and Phase II solutions to a        flow-focusing junction;    -   (v) Encapsulation of Phase I and Phase II in droplets suspended        in carrier oil;    -   (vi) Droplet collection off-chip.    -   (vii) The species are provided with first fluid, with second        fluid or with both.

The term “Phase I solution”, as used herein, refers to a solution thatis miscible with Phase II solution, but it can form a separate phaseduring so called liquid-liquid phase separation process, which occurspassively or upon external force (e.g. gravity). Similarly, the term“Phase II solution”, as used herein, refers to a solution that ismiscible with Phase I solution, but can form a separate phase duringliquid-liquid phase separation process.

In one aspect, Phase I solution is rich in Dextran.

In another aspect, Phase II solution is rich in a polymer based onpolyethylene glycol.

In one exemplary embodiment, the droplet generation occurs atcross-junction having a nozzle (constriction) where the break-up offluid stream into monodisperse droplets occurs.

In another aspect, the invention comprises the method in which the phaseseparation of Phase I and Phase II solutions occurs in liquid droplets.

In another aspect, the invention comprises the method in which the PhaseI and Phase II solutions form inner phase and outer phase in liquiddroplets.

In yet another aspect, the invention comprises the method in which thePhase II is hardened by triggering a polymerization.

Polymerization (gelation) herein, refers to the process in which aliquid form of “Phase II solution” is forming a solid or semi-soldhydrogel in the contact with “inducer”. Typically, but not limited to,inducer can be light, chemical compounds, temperature, etc.

In yet another aspect, the invention comprises the method in whichcapsules are released from droplets by breaking the emulsion.

In yet another aspect, the invention comprises the capsules composed ofsemi-permeable shell and liquid-like core.

In one exemplary embodiment, the cells, biochemical and biologicalcompounds are introduced in droplets by supplying them:

-   -   (i) In the “Phase I solution”;    -   (ii) In the “Phase II solution”;    -   (iii) In all solutions.

In one aspect the invention describes the use of capsules for processingencapsulated species in multi-step sequential operations.

In another aspect the invention describes the use of capsules forperforming multi-step reactions on encapsulated species.

In another exemplary embodiment, encapsulated species are cells that arelysed inside the capsules.

In another exemplary embodiment, the reagents that were used to lyse thecells are replaced by suspending capsules in a different buffer.

In yet another exemplary embodiment, the buffer in which cells werelysed is replaced with another buffer by suspending capsules in a saidbuffer.

In one exemplary embodiment, the encapsulated species are processed inmulti-step sequential operations to perform a desirable biochemical orbiological reaction.

In one exemplary embodiment, said reaction can be DNA amplificationwhere the nuclei acids of lysed cells are amplified enzymatically usingphi29 DNA polymerase.

In another exemplary embodiment, the nuclei acids of lysed cells areamplified enzymatically by PCR.

In another exemplary embodiment, the encapsulated cells are maintainedalive over extended periods of time.

In another exemplary embodiment, the encapsulated cells are cultivatedover extended periods of time.

In another specific embodiment, the individual cells are expanded intomicrocolonies.

In one specific exemplary embodiment, the encapsulated cells arescreened for biological activity (e.g. production of metabolites,proteins, compounds, etc.)

In another exemplary embodiment, the phenotypic and/or genotypicanalysis is performed on encapsulated cells and/or their material.

In one exemplary embodiment, the above methods are carried out but notlimited to using a microfluidics system.

In one exemplary embodiment, the capsules have a size ranging fromapproximately 20 to 100 μm.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Concentric capsule production and characterization. (a) Capsuleformation using co-flow microfluidic device. (b) Liquid-liquid phaseseparation inside double emulsion droplets resulting in Dextran-richcore and PEGDA-rich shell. (c) Double emulsion droplets from panel (b)imaged under fluorescence microscope. Droplets contained 0.1% (w/v)fluorescein isothiocyanate-Dextran (MW 500K). (d) Capsules recoveredfrom emulsion, where PEGDA shell formed firm hydrogel and Dextran phaseformed liquid-like core. (e) Histograms, derived from N>100measurements, indicate capsule shell thickness, capsule core and overalldiameter. Scale bars, 50 μm.

FIG. 2. Concave versus core-shell capsule formation. In panel (a)capsules were prepared using the composition of 6% PEGDA (MW 8K) and 3%PEGDA (MW 575), while in panel (b) 3% PEGDA (MW 8K) and 3% PEGDA (MW575) were used. (c) Boxplots demonstrate the difference in densitybetween PEGDA and Dextran phases. Scale bars, 50 μm, boxplots arederived from samples of N>25 measurements.

FIG. 3. Capsule production. Panel (a) shows PEGDA and Dextran phaseseparation after centrifugation, where Dextran phase was stained with0.1% (w/v) fluorescein isothiocyanate-Dextran (MW 500K). (b) Separationof two phases after photo-polymerization, where PEGDA-rich phase formedhardened hydrogel shell and Dextran-rich phase became more viscous. (c)ATPS droplets showing dextran phase partitioning into the core. Panels(d) and (e) show capsules under bright field and fluorescent microscope,respectively, whereas (f) is merged images. Scale bars, 50 μm.

FIG. 4. Comparison of multi-step processing of encapsulated E. colicells in three microfluidic formats: 1) droplets (water-in-oilemulsion), 2) hydrogel beads and 3) capsules. On the left, merged imagesshowing fluorescence intensity of each assay before and after cell lysisand after MDA reaction. Following exposure times were used forfluorescent microscopy: before and after lysis—1 s, after MDA—10 ms.Bacteria cells, suspended in 10 mM Tris-HCl [pH 7.5] wereco-encapsulated with Lysozyme, Triton X-100, Phi29 DNA Polymerase bufferand DTT (see Experimental Methods). E. coli containing hydrogel beadswere produced with 6% (v/v) PEGDA (MW 575), whereas for capsulesproduction the blend contained 3% (w/v) PEGDA (MW 8K), 3% (v/v) PEGDA(MW 575) and 5.5% (w/v) Dextran (MW 500K) was taken. To visualize DNA,the samples were stained with 1×SYBR Green I dye and analyzed underfluorescent microscope using the following exposure times: before andafter lysis—1 s, and after MDA—10 ms. On the right, boxplotsrepresenting mean fluorescence intensity in positive and empty post-MDAcompartments. Boxplots were derived from N>500 measurements, stars showstatistical significance based on t-test. Scale bars, 50 μm.

FIG. 5. Lambda value (occupancy) measurements of E. coli encapsulation,lysis and MDA reaction in three microfluidic formats: 1) water-in-oilemulsion (green), 2) hydrogel beads (blue), and 3) capsules (red).Boxplots are derived from samples of N>500 measurements. Note, hydrogelbeads showed a significant drop in lambda value after lysis step.

FIG. 6. E. coli distribution inside hydrogel beads and capsules. (a)Partition of encapsulated E. coli cells in hydrogel beads, where bluearrows indicate bacteria close to the interphase. (b) Partition ofencapsulated E. coli cells in capsules, where red arrows indicatebacteria close to the Dextran-PEGDA interface. Scale bars, 50 μm.

FIG. 7. Comparison of MDA reaction yield on single B. subtilis inwater-in-oil emulsion and in capsules. Merged fluorescence and brightfield images of (a) post-MDA droplets and (b) post-MDA capsules. (c)Comparison of MDA reaction efficiency based on fluorescence intensity incapsules (red) and in droplets (green). Boxplots are derived fromsamples of N>500 measurements, stars show statistical significance basedon t-test. Scale bars, 50 μm.

FIG. 8. Post-MDA capsule analysis by flow cytometry. Histogram shows150.000 capsule fluorescence distribution, where the intensity <6.1represents negative and >6.1 positive capsules.

FIG. 9. Analysis of PCR amplicon retention inside capsules. Above,merged images showing the capsules after the synthesis of differentlength PCR amplicons: ompA (1050 bp), kdsC (567 bp), 16S rRNA (320 bp).Below, boxplots, representing lambda value (the mean number of bacteriain each capsule following Poisson distribution) before and after celllysis, and after PCR with corresponding PCR amplicons. Histograms arederived from samples of N>500 measurements. Scale bars, 50 μm.

FIG. 10. DNA recovery from post-PCR and post-MDA capsules by alkalinetreatment. 5 μl of capsules, harboring MDA product or one of threeamplicons (320, 567 or 1050 bp.) were dissolved in 1M NaOH, neutralizedwith 1M acetic acid, and released DNA was purified/concentrated usingAMPureXP magnetic beads. The expected size of ompA, kdsC and 16S RNAamplicons is 1050, 567 and 320 bp, respectively. M—GeneRuler DNA LadderMix (SM0331).

FIG. 11. E. coli (MG1655) cultivation in capsules with the compositionof 3% (w/v) PEGDA (MW 8K), 3% (v/v) PEGDA (MW 575) and 5.5% (w/v)Dextran (MW 500K). The cell growth was monitored for 8 hours. Sampleswere analyzed by brightfield and fluorescence microscopy after stainingwith 1×SYBR Green I. Exposures times: 0 h—400 ms, 4 h-8 h—10 ms. Scalebars, 50 μm.

FIG. 12. Comparison of E. coli (MG1655) bacteria growth in capsules andin droplets. Panels (a) and (d) show capsules and droplets at 0 h, (b)and (e)—after 4 h of cell culture at 37° C. Panels (c) and (f) representfluorescent bacteria from images (b) and (e) after staining with SYBRGreen I dye. (g) Boxplots, representing bacteria counts after 4 hours ofincubation. Exposure time for (c) image was 10 ms, and for (f) −100 ms.Boxplots are derived from samples of N>20 measurements, stars showstatistical significance based on t-test. Scale bars, 50 μm.

FIG. 13. Normalized fluorescence of positive droplets and capsulescontaining E. coli colonies after 4 hours growth. Boxplots are derivedfrom samples of N>500 measurements, stars show statistical significancebased on t-test.

FIG. 14. Phenotypic analysis of PHB-producing micro-colonies based onNile Red staining. Live micro-colonies of bacteria producing PHB (a andc) and negative control (b and d) showing no significant difference influorescence. Lysed micro-colonies of PHB-producing clones (e) andnegative control (f) after washing the capsules to remove solubilizedmembranes. (g) Boxplot showing how cell lysis and capsule washingincreased the ability to resolve PHB-producing colonies from negativecontrols. Exposure times for (c)-(f) images were 100 ms. Boxplots arederived from samples of N>100 measurements, stars show statisticalsignificance based on t-test. Scale bars, 50 μm.

FIG. 15. Evaluation of bacterial metabolite PHB synthesis followingsingle-cell derived colony expansion and lysis. Left: capsules withpositive (DH5α-pBHR68) and negative (DH5α-pTZ18R) bacteria. Bright fieldimages show lysed micro-colonies, Nile Red staining shows PHB metaboliteamount, SYBR I indicates bacteria quantity in individual capsules basedon gDNA staining, and Merged image shows Nile Red and SYBR I imagescombined. Lysed bacteria were stained with Nile Red and SYBR Green I tonormalize PHB levels to bacteria count. Scale bars, 50 μm. Right:boxplots are derived from samples of N>100 measurements, stars showstatistical significance based on t-test.

FIG. 16. Evaluation of reverse transcription and PCR reaction onmammalian cells using capsules. Bright-field and fluorescencemicroscopy, and merged images of mammalian cells in capsules afterlysis, DNAse I treatment, RT-PCR and PCR (without RT) are shown. Sampleswere stained with SYBR Green I dye. Scale bars, 100 μm.

FIG. 17. DNA recovery and analysis of PCR and RT-PCR product formationon capsule-encapsulated mammalian cells. The results in “A” gel wereobtained performing cell lysis, while the results in “B” gel—applyingboth cell lysis and gDNA elimination steps before PCR and RT-(PCR),respectively.

A gel:

Well-1, M—GeneRuler DNA Ladder Mix (SM0331);

Well-2, SRSF1 amplicon obtained by performing PCR;

Well-3, SRSF1 amplicon obtained by performing PCR;

Well-4, Negative control: —PCR enzyme using the same capsules as above.

B gel:

Well-1, M—GeneRuler DNA Ladder Mix (SM0331);

Well-2, Negative control: no RT step (only PCR) using the same capsulesas in 3th sample;

Well-3, ACTB amplicon obtained by performing RT-PCR.

SRSF1 product (˜500 bp) and ACTB product (˜700 bp), marked with red andgreen squares, respectively, were obtained after DNA extraction from ˜10μL close-packaged capsules. M—GeneRuler DNA Ladder Mix (SM0331).

FIG. 18. Occupancy (lambda value) measurements at different stages ofencapsulated cell processing. Green, blue, red and orange boxplots showlambda value in water-in-oil droplets, in capsules after lysis, incapsules after DNAse I treatment and in capsules after RT-PCR.

FIG. 19. Other examples of semi-permeable capsules. Left: capsulesproduced using PEGDA-Citrate system. Right: capsules produced usingPEGDA-PVA system. Scale bars, 50 μm.

FIG. 20. Example of hydrogel capsules whose shell can be dissolved withreducing agents such as 10 mM DTT. Scale bar, 100 μm.

FIG. 21. Example of hydrogel capsules (whose shell can be dissolved withreducing agents) use to perform multi-step operations and reactions onencapsulated species. Capsules before E. coli lysis (the left picture),after E. coli lysis (the middle picture) and capsules after PCR withkdsC primers being used (the right picture).

FIG. 22. Agarose gel of PCR product released from capsules. M—GeneLulerDNA ladder Mix. Lane 1—PCR product using kdsC primers, Lane 2—PCRproduct using ompA primers and Lane 3—negative control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to multi-step processing ofcapsule-encapsulated species to perform desired biological orbiochemical reaction(s). In this context the capsules provide thesemi-permeable compartment (reactor) for processing encapsulated speciesthrough multiple chemical conditions. The capsules may be used forencapsulation of cells, viruses, DNA and/or other biological compounds.In some cases, the capsules may be used in biological or biochemicalassays. In some other cases, the present invention relates toalternative solutions to a particular problem, and/or a plurality ofdifferent uses of one or more systems and/or methods.

In one aspect the invention is a method for multi-step processing ofcapsule-encapsulated species. The present invention provides a methodfor forming/providing a fluidic droplet containing the species, causinga separation into inner and outer phases of the fluidic dropletcontaining the species, inducing the gelation of the outer phase of thefluidic droplet containing the species, and performing reaction(s)and/or analysis on the encapsulated species.

In another aspect the invention is a method for encapsulation ofbiochemical and biological compounds, cells, viruses, DNA and othermolecules to perform desired biochemical or biological reaction onencapsulated species. Once the capsule is formed and species areisolated, the composition of the capsules can be changed by adding newreagents or replacing out old ones (e.g. by capsule resuspension indesired solution).

In another aspect the invention is a method for performing multi-stepoperations on encapsulated entities. The encapsulated entities (e.g.cells, DNA, etc) can be retained inside the capsules or released fromthem upon external stimulus as deem desirable.

In the method of the invention, the encapsulated species are exposed todifferent chemical conditions in a sequential manner in order to performa desirable reaction on encapsulated species.

In the method of the invention, the microfluidics chip comprises, butnot limited to, following units:

-   -   (i) an inlet and microfluidic channel(s) for carrier oil;    -   (ii) an inlet and microfluidic channel(s) for the first fluid;    -   (iii) an inlet and microfluidic channel(s) for the second fluid;    -   (iv) a nozzle or cross-junction;    -   (v) a microfluidics channel connecting the nozzle with the        outlet, and    -   (vi) collection outlet.

In another aspect, the invention comprises the method for the formationof liquid/fluid droplets using microfluidics chip for:

-   -   (i) Injection of a “Phase I solution”;    -   (ii) Injection of an “Phase II solution”;    -   (iii) Injection of a carrier oil;    -   (iv) Brining carrier oil, Phase I and Phase II solutions to a        flow-focusing junction;    -   (v) Encapsulation of Phase I and Phase II solutions in droplets        suspended in carrier oil;    -   (vi) Droplet collection off-chip.

The term “microfluidic chip”, as used herein, refers to a device, orchip, of only millimetres to a few square centimetres or tens ofcentimetres in size dealing with the handling of extremely small fluidvolumes down to less than picoliters. Microfluidic chips are usuallyfabricated by using lithography-based technologies such as softlithography.

In an embodiment, the fluids are introduced into the microfluidics chipvia an inlet(s) and pass through the passive filter(s) and/or fluidresistor(s).

In a more particular embodiment, passive filters used in the chip of theinvention are used to prevent microfluidic channels from clogging andact as solid support to avoid collapse of device structure. The fluidresistors damp fluctuation that might arise during device operation.These units may be well-known by the skilled person and their uses areillustrated in FIG. 1.

In an embodiment, the micro-channels of each fluid are merging into asingle micro-channel upstream the flow-focusing junction whereindividual fluids meet but do not mix (FIG. 1).

In an embodiment, the depth of the microfluidic channels are in therange from 1 μm to 100 μm, preferably in the range 10-40 μm.

In the method of the invention, the droplet generation occurs atcross-junction having a nozzle (constriction) where the break-up offluid stream into monodisperse droplets occurs.

In the method of the invention, the Phase I and Phase II solutions areinjected to microfluidics chip and form fluid droplets.

The term “Phase I”, as used herein, refers to a solution that ismiscible with Phase II solution, but it can form a separate phase duringso called liquid-liquid phase separation process, which occurs passivelyor upon external force (e.g. gravity). Similarly, the term “Phase II”,as used herein, refers to a solution that is miscible with Phase Isolution, but can form a separate phase during liquid-liquid phaseseparation process.

In an embodiment, Phase I solution is rich in Dextran.

In an embodiment, Phase II solution is rich in modified polyethyleneglycol polymer that can be cross-linked.

In another aspect, the phase separation occurs in droplets (FIG. 1).

In another aspect or the invention comprises the Phase I and Phase IIsolutions form inner phase and outer phase in liquid droplets,respectively.

In the method of the invention, the Phase I and Phase II solutions canbe selected from a range of polymer systems available, as described inJ. M. S. Cabral, Cell Partitioning in Aqueous Two-Phase Polymer Systems,Adv Biochem Engin/Biotechnol (2007).

In the method of the invention, the Phase II solution is hardened byinducing a polymerization (e.g. using light). Polymerization (gelation)herein, refers to the process in which a liquid form of “Phase IIsolution” is forming a solid or semi-sold hydrogel in the contact with“inducer”. Typically, but not limited to, inducer can be light, chemicalcompounds, temperature, etc.

In the method of the invention, once the capsule is formed itscomposition can be changed by adding new reagents or replacing out oldones.

In the method of the invention, the liquid droplets are collectedoff-chip via outlet.

In an embodiment, the droplets are generated on the microfluidic chipcomprising a flow-focusing junction (as illustrated in FIG. 1) allowingthe production of droplets of different size. The droplet size can becontrolled by adjusting the flow rates of Phase I and Phase solutionsand carrier oil and/or the cross-section of a nozzle and/or thecross-section of microfluidics channels.

In an embodiment, the droplets are generated at a frequency ranging from0.01 Hz to 10 kHz, preferably from 0.1 kHz to 5 kHz, more preferablyfrom 0.5 kHz to 2.5 kHz. A frequency of 1 kHz means that droplets areprovided at a rate of 1000 droplets per second.

As used in this specification, the term “about” refers to a range ofvalues ±10% of the specified value. For example, “about 20” includes±10% of 20, or from 18 to 22. Preferably, the term “about” refers to arange of values ±5% of the specified value.

In an embodiment, the droplets have a volume ranging from 0.01 pL to1000 nL, preferably from 100 pL to 500 pL and more preferably from 10 pLto 100 μL.

In a particular embodiment, Phase I, Phase II or both solutions maycomprise, for instance, various chemical compounds such as buffers,salts, carbohydrates, lipids, polymers, proteins, nucleic acids, cellsor micro-organisms.

In a particular embodiment, the carrier oil used to generate droplets isa fluorinated oil and comprises a surfactant, a PFPE-PEG-PFPE(perfluoropolyether-polyethylene glycol-perfluoropolyether) tri-blockcopolymer. Said surfactant being present in the carrier oil at aconcentration ranging from 0.05% to 3% (w/w), preferably ranging from0.1% to 1% (w/w), more preferably ranging from 1% to 3% (w/w).

The method of the present invention is not limited by the type ofsurfactant or carrier oil used. One of ordinary skill in the art will beable to select the appropriate surfactant, dispersed phase and carrieroil based on the desired properties of the droplets and reactionconditions used.

Surfactants, also named emulsifying agents, act at the water/oilinterface to prevent (or at least to decay) separation of the phases.

In an embodiment, the carrier oil is selected from the group consistingof fluorinated oil such as FC40 oil (3M®), FC43 (3M®), FC77 oil (3M®),FC72 (3M®), FC84 (3M®), FC70 (3M®), HFE-7500 (3M®), HFE-7100 (3M®),perfluorohexane, perfluorooctane, perfluorodecane, Galden-HT135 oil(Solvay Solexis), Galden-HT170 oil (Solvay Solexis), Galden-HT110 oil(Solvay Solexis), Galden-HT90 oil (Solvay Solexis), Galden-HT70 oil(Solvay Solexis), Galden PFPE liquids, Galden® SV Fluids or H-Galden® ZVFluids; and hydrocarbon oils such as Mineral oils, Light mineral oil,Adepsine oil, Albolene, Cable oil, Baby Oil, Drakeol, ElectricalInsulating Oil, Heat-treating oil, Hydraulic oil, Lignite oil, Liquidparaffin, Mineral Seal Oil, Paraffin oil, Petroleum, Technical oil,White oil, Silicone oils or Vegetable oils. In a particular embodiment,the carrier oil is a fluorinated oil. In a more particular embodiment,the carrier oil is HFE-7500 oil.

In a preferred embodiment, the depth of all channels on the microfluidicchip is the same and is in the range from 1 μm to 1000 μm, preferably inthe range 50-500 μm and more preferably in the range of 20-300 μm, andeven more preferably in the range of 10-100 μm.

In a further embodiment, the method of the invention further comprisescollecting fluid droplets off-chip.

In another embodiment, the collected droplets are broken therebyreleasing capsules into surrounding media. This can be achieved bydestabilizing the droplet water-oil interface using chemical means orusing electro-coalescence, temperature, dilution, etc. In thisparticular embodiment, the droplet water-oil interface is destabilizedby mixing the emulsion with chemical such as fluorinated octanol.

In one exemplary embodiment, the capsules are composed of semi-permeableshell and liquid-like core.

In a preferred embodiment, the semi-permeable shell is a hydrogelcomposed of PEGDA, and liquid-like core is enriched in Dextran.

In one exemplary embodiment, the cells, biochemical and biologicalcompounds are introduced into liquid droplets/capsules by supplying thesaid entities in:

-   -   (i) In the “Phase I solution”;    -   (ii) In the “Phase II solution”;    -   (iii) In both solutions.

In the method of the invention, the species encapsulated in capsules areexposed to different biochemical environment by suspending the saidcapsule in desired aqueous solution or polar solvent.

In another aspect of the invention, the species encapsulated in capsulesare reacting with chemical, biochemical or biological compounds presentin aqueous solution.

In another exemplary embodiment, the encapsulated cells are lysed insidethe capsules.

In another example, the material of lysed cells is fully or partiallyretained inside the capsules.

In yet another example, the reagents that were used to lyse theencapsulated cells are replaced by suspending capsules in a differentaqueous solution.

In one exemplary embodiment, the nuclei acids of lysed cells areamplified enzymatically. In one specific embodiment the nucleic acidsare amplified using phi29 DNA polymerase, yet in another exemplaryembodiment the nuclei acids of lysed cells are amplified enzymaticallyby PCR. Obviously, other enzymes (e.g. Klenow, Bst, Bsm polymerases) arealso possible to use for nucleic acid analysis, replication andamplification.

In one exemplary embodiment, the mRNA of lysed cells is converted tocDNA by reverse transcription reaction. In one specific embodiment thecells encapsulated in capsules are lysed and their mRNA is converted tocDNA using reverse transcriptase.

In the method of the invention DNA and/or RNA of lysed cells can bemodified/treated using chemical or biochemical means. For example, addpoly(A) tail to nucleic acids, add nuclei acid barcodes, add indexes,ligate adapters, digest, fragment, etc.

In the method of the invention the cDNA of encapsulated individual cellscan be tagged (barcoded) with barcoded poly(T) primers.

In the method barcoded poly(T) primers can carry cell barcode, molecularbarcode (unique molecular identifier), sequencing adapter, poly-dT partand/or other parts as required for barcoding reaction.

In another exemplary embodiment the cDNA of lysed cells is amplifiedenzymatically by PCR inside or outside the capsules.

In the method of the invention the lysis and nucleic acid amplificationis performed on the same encapsulated cells by performing sequentialmulti-step reactions.

In one preferred embodiment the capsules are used for genotypic analysisof individual cells.

In yet another preferred embodiment, the encapsulated cells aremaintained alive over extended periods of time to perform phenotypicanalysis of individual cells.

In another exemplary embodiment, the encapsulated cells are cultivatedover extended periods of time. In one specific exemplary embodiment, theencapsulated cells are screened for biological activity (e.g. metabolicactivity).

In one exemplary embodiment, the biochemical and biological moleculesentrapped inside the particles are released by dispersing particles intoa surrounding fluid such as biological buffer, water and/or otheraqueous solutions.

In exemplary embodiment, the phenotypic and/or genotypic analysis isperformed on encapsulated cells and/or their material.

In exemplary embodiment, the above methods are carried out but notlimited to using a microfluidics system. The microfluidic system may beinstalled by the skilled person.

The following examples of the invention is given for purposes ofillustration and not by way of limitation.

EXAMPLES

Experimental Methods

Materials and Reagents

Device fabrication and operation. The polydimethylsiloxane (PDMS)microfluidic device was fabricated and operated using standardizedprotocol as described (Mazutis et al., Nature Protocols, 2017).Preparation of ATPS. All chemicals were ordered from Sigma-Aldrich andFisher Scientific. ATPS droplets were prepared using 5.5% (w/v) Dextran(MW 500K), 3% (w/v) PEGDA (MW 8K), 3% (v/v) PEGDA (MW 575), 0.1% (w/v)LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate), 1×DPBS. Modifiedconcentrations of PEGDA (MW 8K) and PEGDA (MW 575) as well as modifiedpolymers could be used. The solutions containing all ingredients weremixed and centrifuged in a table centrifuge at maximum speed for 30minutes to induce liquid-liquid phase separationPreparation of hydrogel beads. The mixture of 6% (v/v) PEGDA (MW 575)and 0.1% (w/v) LAP in 1×DPBS was used for hydrogel bead preparation andbacteria embedding following hydrogel bead production protocol aspreviously described (Mazutis et al., Nature Protocols, 2017).Preparation of microbial cells. Escherichia coli (MG1655 and DH5α),Bacillus subtilis (SHgw) as well as pBHR68 and pTZ18R plasmids werekindly provided by Prof. R. Meškys (Vilnius University, Institute ofBiochemistry, Lithuania). DH5α strain was transformed with pBHR68plasmid, harboring three genes (phaC, phaA and phaB) from PHB synthesispathway. As a negative control, DH5α strained transformed with pTZ18Rvector was used. Prior the encapsulation bacteria were suspended inDextran-rich phase and when needed supplemented with 100 μg/mLAmpicillin.

Emulsification. As exemplified in FIG. 1 droplets, hydrogel beads andcapsules were generated using microfluidics chip 20 μm height and havinga nozzle 15 μm wide. Typical flow-rates used were: PEGD(M)A-rich phase−50 μL/hr, Dextran-rich phase or 1×DPBS (with/without bacteria) −50μL/hr and droplet stabilization oil (Droplet Genomics, DG-DSO-20) −350μL/hr. For larger size capsule generation 50 μm height microfluidicschip was used with the following flow rates: PEGD(M)A-rich phase −140μL/h, Dextran-rich phase with cells −70 μL/h and droplet stabilizationoil (Droplet Genomics, DG-DSO-20) −600 μL/h. Due to increased viscosityof biphasic system, droplet breakup by jetting mechanism could beobserved, which could shift to dripping mode by adjusting the flow ratesof a system.

Cross-linking. Emulsions were collected in a 1.5 ml tube and immediatelycross-linked by exposure under 365 nm wavelength using High-Intensity UVInspection Lamp, UVP (UVP, 95-0127-01) for 2.5 minutes. ATPS dropletsfor bacteria culture experiments were exposed to 405 nm laser (1 W/cm²)for 20 seconds. After hardening the PEGDA shell resulting capsules wererecovered from the emulsion using commercial emulsion breaker (DropletGenomics, DG-EB-1).Lysis and DNA amplification in hydrogel beads and capsules. Lysis ofencapsulated bacteria was performed by suspending hydrogel beads orcapsules in lysis buffer containing: 50 U/μL Ready-Lyse™ LysozymeSolution (Lucigen, R1804M), 200 μg/mL Proteinase K (Invitrogen, AM2546),0.1% (v/v) Triton X-100 (Sigma-Aldrich, T8787-100 ML), 10 mM Tris-HCl[pH 7.5] and 1 mM EDTA. Hydrogel beads and capsules suspended in lysisbuffer were incubated for 30 min at 37° C. followed by additional 30 minincubation at 50° C. After lysis, hydrogel beads and capsules werewashed three times in a Washing buffer (10 mM Tris-HCl [pH 7.5] and0.05% (v/v) Triton X-100). MDA reaction was then performed by suspendingcapsules and hydrogel beads in MDA reaction buffer containing 0.5 U/μLphi29 DNA polymerase (Thermo Scientific, EP0092) and 0.002 U/μLinorganic pyrophosphatase (Thermo Scientific, EF0221) followingmanufacturer's recommendations. Bulk-like PCR was used to amplifyspecific regions of 16S rRNA, kdsC and ompA genes corresponding to 320,567 and 1050 bp fragments, respectively. Each amplification wasperformed for 35 cycles with KAPA PCR kit (KAPABiosystems, KK2602)according to manufacturer's recommendations. In all enzymatic reactions,the close-packaged capsules and hydrogel beads occupied approx. 40-50%of the final reaction volume.Lysis and DNA amplification in droplets. To perform E. coli and B.subtilis lysis in droplets, bacteria were re-suspended in 10 mM Tris-HCl[pH7.5] and co-encapsulated with Ready-Lyse™ Lysozyme Solution, TritonX-100, phi29 DNA polymerase buffer and DTT at the final concentration of50 U/μl, 0.1% (v/v), 1× and 1 mM, respectively. When cell lysis and MDAreaction was performed simultaneously, the final reaction compositionwas: 1× Reaction Buffer for phi29 DNA polymerase, 25 μM Exo-resistantrandom primer (Thermo Scientific™, S0181), 1 mM dNTP Mix (ThermoScientific™, R0192), 1 mM DTT (Thermo Scientific™, 707265ML), 0.1% (v/v)Triton X-100 (Sigma-Aldrich, T8787-100ML), 50 U/μl Ready-Lyse™ LysozymeSolution (Lucigen, R1804M), 0.5 U/μl phi29 DNA polymerase (ThermoScientific, EP0092) and 0.002 U/μl inorganic pyrophosphatase (ThermoScientific, EF0221). The encapsulation conditions as well as MDAreaction conditions were the same as with capsules.Imaging of processed bacteria. Droplets, hydrogel beads and capsuleswere stained with 1×SYBR Green I dye (Invitrogen, S7563) and analyzedunder inverted fluorescence microscope using the following settings:magnification—10×, filter—FITC, gain—1, 20% intensity of blue lightsource used for excitation and exposure time were varied depending onthe analysis step. Images were recorded using digital camera (Nikoneclipse Ti at 12-bit resolution).Capsule analysis by flow cytometry. The capsules were stained with1×SYBR Green I dye and analysed on Sapphire microfluidics platform(Droplet Genomics, DG-SPH-1). A total of 150.000 capsules were measuredusing a 488 nm diode laser (1 mW) focused to a 40 μm diameter channel.Capsule solubilization and DNA extraction. Capsules were dissolved inthe presence of 1 M NaOH at 50° C. for 10 minutes and then neutralizedby adding equimolar amount of 1M Acetic Acid. PCR products fromdissolved capsules were extracted and concentrated using 1.8× AgencourtAMPure XP magnetic beads (Beckman Coulter, A63881) and analyzed on 1%agarose gel.Bacteria cultivation inside capsules. All bacteria growth experimentswere performed in disposable 30×15 mm Petri dishes. MG1655 bacteriaencapsulated in capsules, hydrogel beads or droplets were cultivated inLB medium at 37° C. for 4-8 h, while the media for transformed DH5αstrain was supplemeted with 100 μg/mL Ampicillin. After reachingexponential growth (4-6 h), polyhydroxybutyrate (PHB) synthesis in DH5αwas induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (ThermoScientific, R1171) followed by incubation at 30° C. for 8 h.Imaging of encapsulated bacteria. Capsules and droplets with MG1655cells were stained with 1×SYBR Green I dye and analyzed under invertedfluorescence microscope. DH5α strain was stained for 10 minutes withNile Red (0.5 μg/mL) and analyzed using the following settings:magnification—10×, filter—TXRED, gain—1, exposure time −100 ms, 40%intensity of green light source for excitation. The second round ofimaging was performed after lysis (without the additional staining withNile Red) using the same conditions to evaluate the changes offluorescence. For dual DH5a imaging, capsules were stained repeatedlywith Nile Red and SYBR Green I dyes. Images were taken using followingsettings: magnification—1×, filters—FITC and TXRED, gain—1, exposuretime—10 ms for FITC filter and 40 ms for TXRED, 20% and 40% intensity ofblue and green light source for excitation, respectively. Images wererecorded using Nikon eclipse Ti camera at 12-bit resolution on aninverted fluorescence microscope.Data processing. Fluorescence data was obtained by manually outliningdroplets from brightfield images and then using these masks to segmentfluorescence images. Data was managed and analyzed using R (v.3.5.3) andR studio (v.1.1.463). Capsule fluorescence was normalized to imagebackground and image acquisition settings were kept the same duringcomparative experiments. Fluorescence was reported as logarithmic valuesto control the dynamic range, normalize dispersion and allow comparingdim and bright objects. Positive/Negative capsule identification wasachieved based on fluorescence data histogram analysis. T-testing wasused for statistical significance measurements, where stars indicatep-value ranges: *(0.05-0.01), **(0.01-0.001), ***(P<0.001).

Results

In the current state-of-the-art one of the biggest challenges forimplementing capsule formation based on aqueous two-phase system (ATPS)is an inconsistent and non-uniform shell formation (Ma, S. et al. Small8, 2356-2360 (2012). Mytnyk et al., RSC Adv., 2017, 7, 11331-11337). Wehave noticed that the capsule core tends to migrate towards the outerinterphase before shell gelation could occur, leading to a concaveparticle topology. As a result, a significant fraction of capsulesreleased their encapsulated material prematurely, contained uneven orruptured shells (FIG. 2). Obviously, such defective capsules wouldhinder many biological applications that mandate efficient sampleencapsulation, retention and processing reproducibility. Since thecapsule's shell uniformity depends on concentricity of ATPS droplets, wepostulated that the density mismatch between the core and shell phaseswas driving the core of-centre. We show that by reducing the densitydifference between the two aqueous phases enables consistent generationof monodisperse and concentric ATPS droplets (FIGS. 1 and 2). Tosolidify the shell of ATPS droplets en masse we used photo-illumination,as a chemically neutral measure to induce fast (˜2 min) PEGDApolymerization and form a hardened shell. The resulting capsulescontained aqueous (liquid-like) core enriched in Dextran phase andsolidified hydrogel-shell, enriched in PEGDA. We noticed that the coreof capsules becomes more viscous after photopolymerization (FIG. 3)presumably due to the presence of residual PEGDA and/or formation of aweak hydrogel mesh, however that did not have a negative effect onbiological assays presented herein.

To achieve the right balance between capsule uniformity, concentricityand mechanical stability we arrived at the composition containing blendsof longer (MW 8K) and shorter (MW 575) polyethylene glycol diacrylate(PEGDA) polymers, and aqueous Dextran (MW 500K) solution (seeExperimental Methods). Whereas, the longer PEGDA was required forefficient phase separation, the shorter PEGDA was added to increaseshell stiffness and improve capsule mechanical stability. The capsuleswithstood mechanical stress and remained highly uniform with less than2% size variation after washing in aqueous buffer multiple times (FIG.1e ). Furthermore, capsules sustained multiple temperature cycles duringPCR as well as shear forces generated during flow cytometry (see below).In the following we showcase a few examples of semi-permeable capsuleuse in multi-step procedures for genotypic and phenotypic analysis ofindividual bacteria cells in a massively parallel fashion.

Example 1—Nucleic Acid Analysis Using Capsules

Nucleic acid analysis of individual bacterial cells in water-in-oilemulsions can be hindered by the preceding cell lysis step. Chemicalconditions required to break the cell wall of microorganisms caninterfere with downstream reactions, leading to inefficient ornon-uniform DNA amplification, or in case of dropletmicrofluidics—emulsion instability. Bacterial lysis steps can beparticularly problematic for isothermal nucleic acid amplificationmethods (e.g. MDA) or working with gram-positive bacteria, which areknown to be much more resistant to thermolysis. To circumvent abovementioned limitations, the state-of-the-art techniques use hydrogelbeads, where the key feature relies on bacteria embedding into ahydrogel-mesh so that harsh but efficient lysis can be performedseparately from the subsequent enzymatic steps (Spencer, S. J. IMSE. J.,427-436 (2016); Tamminen, M. V. & Virta, M. P. J. Front. Microbiol. 6,1-10 (2015); Novak, R. et al. Angew. Chemie-Int. Ed. 50, 390-395 (2011);Scanlon T. C., et al Biotechnol. Bioeng. 111: 232-243 (2014). Thesehydrogel-bead based systems have convincingly demonstrated thatsemi-permeable carriers can be used for multi-step biochemicalreactions. However, these methods often suffer from complicatedprocessing conditions, including multiple compartment modifications(Tamminen, M. V. & Virta, M. P. J. Front. Microbiol. 6, 1-10 (2015),additional emulsification steps (Spencer, S. J. IMSE. J., 427-436(2016); Novak, R. et al. Angew. Chemie-Int. Ed. 50, 390-395 (2011)) orcomplex microfluidic operations (Lan F., Nat Biotechnol. July;35(7):640-646, (2017)) and more importantly a significant bacteria lossduring hydrogel bead production.

To demonstrate the unique advantages provided by semi-permeable capsulesfor microbiology we compared the efficiency of MDA reaction onindividual E. coli bacterial cells in three different formats; 1)water-in-oil droplets, 2) hydrogel beads and 3) semi-permeable capsules(FIG. 4). For each test, a suspension of E. coli cells was encapsulatedin 10 picoliter (pL) volume droplets together with reagents required forhydrogel bead, or capsule generation (refer to Experimental Methods forfurther details). After encapsulation, the observed occupancy by E. colicells in each assay followed Poisson distribution and was ˜0.2 (FIG. 5)suggesting that there is no significant encapsulation bias betweendifferent droplet assays. However, significant differences emerged afterthe cell lysis step. Whereas the number of positive compartments inwater-in-oil droplets and capsules remained similar, hydrogel-bead basedassay experienced ˜50% cell loss (FIG. 5). Such drastic drop inoccupancy can be explained by bacteria's tendency to localize at theproximity of the water-oil interphase (FIG. 6). As a result, the geneticmaterial released during lysis step becomes susceptible to diffusion outof the hydrogel mesh and eventual loss. Noteworthy, in AIRS droplets thecell partitioning can be controlled by adjusting the ionic species orelectrostatic potential of Dextran-PEGDA phases amongst other components(Cabral, J. M. S., Adv. Biochem. Eng. Biotechnol. 106, 151-171 (2007);Zijlstra, G. M., Systems Biotechnol. Prog., 1996, Vol. 12, No. 3).

Integrated fluorescence measurements showed that the MDA reaction onindividual E. coli cells was 3-times more efficient in capsules ascompared to hydrogel beads (FIG. 4, blue and red boxplots). The higherreaction yield in capsules can be attributed to the liquid-like core,which does not confine the long DNA molecules (>10 kb) synthesized byphi29 DNA polymerase. In hydrogel beads, however, the newly synthesizedDNA is embedded in the hydrogel mesh and is physically confined, thusleading to less efficient replication. Similarly, the MDA reaction yieldin water-in-oil droplets was 2-times higher than in hydrogel-beads (FIG.4, green and blue boxplots), supporting the notion that DNA synthesisreaction is more efficient in a liquid rather than a hydrogel state.Comparing the MDA reaction yield in capsules vs. water-in-oil droplets(FIG. 4, green and red boxplots) we observed mild reaction improvementin capsules, which can be explained by exchange of MDA reactioncomponents through a semi-permeable membrane. In other words, the amountof MDA reagents (dNTPs, primers, enzyme) in capsule-based assay is notconfined by the droplet volume, but can be continuously replenishedthrough a semi-permeable membrane. Capsule advantage over droplets couldbe also explained by more efficient bacteria lysis: the capsules weresuspended in lysis buffer containing Lysozyme and Proteinase K enzymes,while in micro-droplet assay the use of Proteinase K is prohibitive dueto incompatibility with MDA reaction.

We anticipated that the differences in single genome amplificationefficiency will be also pronounced on gram-positive microorganisms whoselysis require harsher conditions that are either inhibitory ordetrimental to subsequent enzymatic steps. To verify this, weencapsulated and subsequently lysed B. subtilis bacteria with a mixtureof Lysozyme and Proteinase K enzymes (the same conditions as used for E.coli). We removed lysis reagents by washing the capsules and thendispersed capsules in the MDA reaction mix to initiate DNA synthesis.The post-MDA capsules were analyzed microscopically (FIG. 7) and by flowcytometry (FIG. 8). As expected, the harsher cell lysis conditions thatwas possible to use with capsules led to approx. 3-times higher MDAreaction yield, when compared to standard conditions using water-in-oildroplets. Furthermore, there was a better separation between positiveand negative compartments (higher signal-to-noise ratio) as well ashigher uniformity of MDA reaction yield (FIG. 7). Combining the MDAresults obtained on B. subtilis and E. coli bacteria we conclude thatcapsules not only efficiently retain the genetic material released uponlysis but more importantly can be processed in a series of enzymaticallyincompatible reactions in order to generate increased yields ofamplified gDNA.

The results presented here convincingly prove that capsules provideefficient physical barrier for retaining the large molecular weightbiomolecules such as bacterial chromosome, or amplified gDNA. To answerthe question, what is the smallest DNA fragment that capsules can stillretain: we generated 320, 567 and 1050 bp. long DNA fragments by PCR andfollowed their diffusion between the compartments. As illustrated inFIG. 9 the capsules efficiently retained 567 bp. DNA fragments, the sizeof which approximately corresponds to MW 340K. Smaller DNA fragments(320 bp. corresponding to approx. MW 190K) diffused between the capsulesas witnessed by appearance of low fluorescence compartments and increasein post-PCR occupancy value. Based on these results and previous reports(Aimar, P., Meireles, M. & Sanchez, V. A. J. Memb. Sci. 54, 321-338(1990)) we estimated the average pore size of the shell to be in therange of 20-50 nm. Finally, to confirm that the expected size DNAfragments were indeed generated during PCR we dissolved the capsules inalkaline solution, extracted DNA and performed electrophoresis. Asexpected, PCR was highly specific and produced expected size DNAfragments (FIG. 10).

Example 2—Cell Cultivation and Phenotypic Analysis Using Capsules

In addition to nucleic acid amplification and analysis (Example 1) manymicrobiology assays also rely on phenotypic characterization. Thiscommonly requires bacterial culture, induced gene expression andsubsequent analysis of proteins or metabolites that serve as aphenotypic readout. Below we demonstrate the use of semi-permeablecapsules for cell culture and screening for metabolic activity incolonies originating from a single bacterium.

We first evaluated whether capsules can be used as micro-chemostats forcell culture applications. For that purpose we encapsulated E. colibacteria using the same microfluidics device as indicated in FIG. 1 andthen immersed the capsules in growth medium for 4 hours at 37° C. Weused time-lapsed microscopy to continuously monitor individual clones,their growth and expansion into isogenic micro-colonies (FIG. 11).Combining this digital data with fluorescence analysis we estimated thatin 4-hours at 37° C. individual clones expanded into micro-coloniescomprising on average 90-cells (FIG. 12). In comparison, cell culture inwater-in-oil droplets generated smaller microcolonies comprising onaverage 30-cells. Furthermore, analyzing bacteria in capsules based onfluorescence signal was considerably easier, as the background from autofluorescent LB media could be removed in a few washing steps, leading toan increased signal-to-noise ratio (FIG. 13).

Having shown the cell growth and isogenic colony formation we thenapplied capsules for phenotypic analysis of bacteria producingpolyhydroxybutyrate (PHB)—an environmentally important biodegradableplastic. Identifying the metabolic products of microorganisms can be achallenge as it may require simultaneous phenotypic (PHB synthesis) andgenotypic (nucleic acid quantification) readouts. To demonstrate thatsuch analysis is possible using semi-permeable capsules we used E. coli(DH5α) strain transformed with pBHR68 vector, harboring genes (phaC,phaA and phaB) for PHB synthesis. We loaded cells in microfluidiccapsules, cultivated them into micro-colonies and then induced PHBsynthesis by adding IPTG. We verified PHB formation in live cell cultureusing Nile Red dye, which stains PHB granules (FIGS. 14a and c ). Beinga lipophilic dye, Nile Red also binds to cell membranes giving a highfluorescent signal in both positive and negative control samples (FIGS.14b and c ). To circumvent the background fluorescence resulting fromnon-specific staining we dissolved cell membranes with lysis reagentsand washed capsules several times greatly increasing the analyticaldifferentiation of positive and negative clones (FIG. 14e-g ).Furthermore, using dual staining, Nile Red dye for PHB granules and SYBRGreen I for gDNA, enabled normalization of PHB levels to bacteria count(FIG. 15), which is crucial for screening efficient producers ratherthan fastest growing clones.

Example 3—Reverse Transcription and Polymerase Chain Reaction inCapsules Mammalian Cell Encapsulation in Capsules

K-562 cells were re-suspend in 100 μL of Dextran-rich phase containing0.4 U/μl RiboLock RNase inhibitor. Final concentration of K-562 cellswas ˜5*10{circumflex over ( )}5/ml. Then ˜100 μm diameter capsules wereprepared using the composition as listed below:

Volume Material Final 10 μl 40% (w/w) PEGDMA (MW 8K) 2% (w/v) 4 μl 100%PEGDMA (MW 550) 2% (v/v) 8 μl 100% PEGDA (MW 575) 4% (v/v) 44 μl 25%(w/v) Dextran (MW 500K) 5.5% (w/v) 4 μl 5% (w/w) LAP 0.1% (w/v) 130 μl1x DBPS — 200 μl Final

Reagents were combined, vortexed and centrifuged at maximum speed for 30minutes. After the separation of two phases (PEGD(M)A and Dextran), thecells were re-suspended in Dextran-rich phase. Encapsulation wasperformed using 50 μm deep microfluidics device. After encapsulationshell polymerization was induced under 365 nm light for 2.5 minutes.Before releasing capsules, 200-300 μl of 1×DPBS+0.3% (v/v) NP-40 wasadded on top of emulsion and then capsules released by breaking emulsionwith 200-400 μl of 20% (v/v) PFO. Capsules were washed several timesfollowed by the lysis step.

Encapsulated Cell Lysis in Capsules

The cell lysis was performed using the composition listed below:

Volume Material Final 30 μl 10% (v/v) NP-40 0.3% (v/v) 10 μl 20 mg/mlProteinase K 200 μg/ml 200 μl Capsules 760 μl 1X DPBS 1000 μl Final

Capsules were incubated in lysis mix at 37° C. for 30 minutes, washed3-4 times with 1×DPBS containing 0.3% (v/v) NP-40 to remove ProteinaseK. Capsules were stained with SYBR Green I and analyzed underfluorescence microscope before proceeding to the next step.

Encapsulated Cell Treatment with DNase I

DNA removal was performed using the reaction composition listed below:

Volume Material Final 50 μl 10χ DNase I Buffer with MgCl2 1X 12.5 μlDNase I, 1 U/μl 0.025 U/μl 5 μl RiboLock RI, 40 U/μl 0.4 U/μl 200 μlCapsules — 232.5 μl Water, nuclease free — 500 μl Final

Capsules were incubated at 37° C. for 20 minutes, washed 3-4 times in1×DPBS containing 0.3% (v/v) NP-40 and fraction of capsules was stainedwith SYBR Green I. Next capsules were processed in reverse transcriptionand PCR reactions.

RT-PCR on Encapsulated Cells

To perform RT-PCR the capsules were suspended in the buffer thosecomposition is listed below:

Volume Material Final 25 μl 2χ Platinum SuperFi RT-PCR Master Mix 1X 2.5μl *ACTB Primer Mix, 10 μM each 0.5 μM 0.5 μl Superscript IV RT Mix — 20μl Capsules — 2 μl Water, nuclease-free — 50 μl Final

As a negative control, samples with no RT enzyme but with 2× PlatinumSuperFi RT-PCR Master Mix was used. The expected size of ACTB ampliconsis 700 bp. To perform RT-PCR reaction following cycling conditions wereused:

Step Temperature Time No. cycles Revere transcription 50° C. 30 min 1 RTinactivation/Initial denaturation 98° C. 2 min 1 Amplification 98° C. 10sec 40 65° C. 10 sec 72° C. 30 sec Final extension 72° C. 1 min 1

After RT-PCR capsules were washed several times with 1×DPBS+0.3% (v/v)NP-40, stained with 1×SYBR Green I and analyzed under fluorescencemicroscope. To dissolve, the capsules were suspended in 1M alkalinesolution incubated at 50° C. for 30 minutes and then neutralized byadding equimolar acetic acid. DNA was extracted and concentrated using1.8× Agencourt AMPure XP magnetic beads (Beckman Coulter, A63881) andanalyzed on 1% agarose gel. Results presented in FIGS. 16-18Bunequivocally prove that capsules are applicable in multi-stepoperations and processes to perform complex biochemical reactions onencapsulated species (e.g. single-cells).

Example 4—Capsules of Different Composition

Using the method and system described here it is possible to generatesemi-permeable capsules composed of different polymers. Below weshowcase two, but not limited to, examples of PEGDA-Citrate andPEGDA-PVA capsules.

TABLE 1 PEGDA-Citrate composition Volume Material Final 16.25 μL 40%(w/w) PEGDA 6.5% (w/v) (MW 8K) 20 μL 40% (w/w) Sodium 8% (w/v) Citrate 2μL 5% (w/w) LAP 0.1% (w/v) 61.75 μL 1x DBPS — 100 μL Final

TABLE 2 PEGDA-PVA composition Volume Material Final 10.5 μL 40% (w/w)PEGDA 4.2% (w/v) (MW 6K) 65 μL 10% (w/w) PVA 6.5% (w/v) (MW 31-50K) 2 μL5% (w/w) LAP 0.1% (w/v) 22.5 μL 1x DBPS — 100 μL Final

After combining the reagents as described in Table 1 or Table 2solutions were mixed well and centrifuge at max speed for 30 minutes.Separate PEGDA and Citrate/PVA phases were loaded onto 20 μm deepco-flow device with the following flow-rates: PEGDA-rich phase −50 μL/h,Citrate/PVA-rich phase −50 μL/h and droplet stabilization oil −350-500μL/h. After encapsulation initiate the polymerization of ATPS dropletsby incubating the tube with emulsion under 365 nm UV lamp for 2.5minutes or 405 nm laser for 20 seconds. Before releasing capsules,200-300 μL of 1×DPBS+0.1% Triton X-100 was added on top of emulsionfollowed by capsule release by adding 200-400 μL of 20% (v/v) PFO.Capsules were washed several times and analysed under the microscope(FIG. 19).

Example 5—Capsules that are Soluble in Some Reducing Environments

Using the method and system described here it is possible to generatesemi-permeable capsules that are sensitive to reducing agents. Below weshowcase, but not limited to, an example of capsules whose shell issensitive to reducing agent. The specific example presented here revealsthat by using a cross-linking agent that is sensitive to reducing agent,it is possible to generate stable semi-permeable capsules that can bedissolved in the presence of reducing agent, typically at concentrationhigher than 1 mM. As referred herein, the reducing agent can bedithiothreitol (DTI), beta-mercaptoehtanol and other compounds ofsimilar nature. To show the example of DTT-soluble capsules, thefollowing reaction mixture was prepared:

Volume Component Final 50 μL 100 mM BAC 50 mM 7.5 μL 40% PEGMEMA, 5K 3%22 μL 25% Dextran, 500K 5.5%   6 μL 100% PEGMA, 360 6% 2.5 μL 40% APS 1%12 μL 1x DPBS — 100 μL Final Note. BAC—(bis(acryloyl)cystamine);PEGMEMA—poly(ethylene glycol) methyl ether methacrylate; PEGMA—poly(ethylene glycol) methacrylate; APS—ammonium persulfate; DPBS—Dulbecco'sphosphate-buffered saline (without calcium and without magnesium).

The reaction mixture was centrifuged at >20.000 RCF for 20 min in orderto form two phases—PEG-rich phase and DEX-rich phase. Each phase wasloaded into a syringe and injected onto microfluidics chip alongsidewith droplet stabilization oil containing 1% TEMED. Using 20 μm deepco-flow device and the following flow rates: droplet stabilization oil400 μL/hr, PEG-rich solution −50 μL/hr and 40 μL/hr for DEX-richsolution, approximately 40 μm size ATPS droplets were collected in theform of an emulsion over 1 hour. Emulsion was incubated at 37° C. for 30minutes to complete hydrogel-shell polymerization. To released hardenedcapsules the emulsion was broken and capsuled resuspended in 10 mMTris-HCl with 0.1% Triton X-100 buffer. After several round of washingthe capsules were analysed under bright field microscope, showingmonodisperse, concentric and nearly-concentric capsules (FIG. 20). Todissolve the capsules, 4 μl of capsule suspension was mixed with 20 μL10 mM DTT and incubated at room temperature for 10, 30 and 60 min. Nocapsules were detected after 10 min proving that produced capsulesremain intact in aqueous buffers but can be dissolved in the presenceof >1 mM DTT.

Example 6—the Use of Capsules, that are Sensitive Reducing Agents, inMulti-Step Procedures

To prove the suitability of capsules (that are sensitive to reducingagents) in the multi-step procedures to perform biological assays weperformed biochemical and enzymatic reactions on encapsulated cells.Following reaction mix was prepared:

Volume Component Final 100 μL 100 mM BAC 50 mM 15 μL 40% PEGMEMA, 5K 3%44 μL 25% Dextran, 500K 5.5%   14 μL 100% PEGMA, 360 7% 5 μL 40% APS 1%22 μL 1x DPBS — 200 μL Final Note. BAC—(bis(acryloyl)cystamine);PEGMEMA—poly(ethylene glyco) methyl ether methacrylate; PEGMA—poly(ethylene glycol) methacrylate; ammonium persulfate; DPBS—Dulbecco'sphosphate-buffered saline (without calcium and without magnesium).

The reaction mixture was centrifuged at >20.000 RCF for 20 min in orderto form two phases—PEG-rich phase and DEX-rich phase. The pellet of E.coli cells was dispersed in DEX-rich phase, loaded into a syringe andinjected onto microfluidics chip alongside with PEG-rich phase anddroplet stabilization oil containing 1% TEMED. Using 20 μm deep co-flowdevice and the following flow rates: droplet stabilization oil 400μL/hr, PEG-rich solution −50 μL/hr and 40 μL/hr for DEX-rich solutioncontaining cells, approximately 40 μm size ATPS droplets were collectedin the form of an emulsion over 1 hour. Emulsion was incubated at 37° C.for 30 minutes to complete hydrogel-shell polymerization. To releasedhardened capsules the emulsion was broken and capsuled resuspended in 10mM Tris-HCl with 0.05% Triton X-100 buffer. After a few rounds ofwashing the capsules were analysed under bright field microscope (FIG.21). The capsules were then suspended in another buffer containing lysisreagents the composition of which is listed below:

Volume Component Final 1.5 μL 33750 U/μL 50.6 U/μL Lysozyme 10 μL 10%Triton X-100 0.1% 10 μL 20 mg/mL 200 μg/mL Proteinase K 100 μL HBs —878.5 μL 1x TE buffer* 1000 μL Final *TE buffer: 10 mM Tris-HCl, 1 mMEDTA, pH 8.

To lyse the cells capsules were incubated in lysis solution at 37° C.for 30 minutes, washed 5-times with 1 mL of 10 mM Tris-HCl containing0.05% Triton X-100 buffer (using 5000 g/2 min centrifugation). Toperform PCR on encapsulated cells following PCR reaction mix wasprepared:

Volume, 1-reaction Component Final 12.5 μL 2x KAPA mix 1x 1 μL 10 μMForward primers mix 0.4 μM 1 μL 10 μM Reverse primers mix 0.4 μM 0.5 μLWater, nuclease-free — 10 μL Close-packaged capsules 25 μL Total Note.kdsC and ompA gene specific primers were used.

After preparing PCR mix samples were incubated in thermo-cycler usingfollowing conditions:

Step Temp Time No Cycles Initial denaturation 95° C. 3 min 1Amplification 98° C. 20 s 35 60° C. 15 s 72° C. 40 s/kb Final extension72° C. 1 min 1 Hold  4° C. ∞ —

To visualize the PCR product in capsules, the capsules were washed3-times with 1 mL Tris-HCl containing 0.05% Triton X-100 and thenstained with 1×Syber Green Dye. Results presented in FIG. 21 show thesuccessful PCR reaction in capsules on single-cells.

To release the PCR product from the capsules, 2 μL of closed packedcapsuels were mixed with 8 μL 10 mM DTT and incubated at roomtemperature for 30 min. The resulting 10 μL were mixed with 2 μL DNALoading Dye (6×) and loaded onto agarose gel followed by theelectrophoresis. Results presented in FIG. 22 show that PCR product ishighly specific, hence unequivocally proving that capsules can withstandmulti-step procedures and are suitable to perform complex reactions, andthat such capsules can be dissolved at desirable time point by addingcertain amount of reducing agent.

1. A method for isolating species in microscopic compartments composedof semi-permeable shell and processing encapsulated species in a seriesof reactions/procedures.
 2. The method according to claim 1, whereinforming/providing a liquid droplet containing the species, causing aseparation into inner and outer phases of the liquid droplet containingthe species, inducing the gelation of the outer phase of the liquiddroplet containing the species, and performing reaction(s) and/oranalysis on the encapsulated species.
 3. The method according to claim2, wherein the outer phase is hardened upon physical or chemicalstimulus (e.g. light, chemical compound, etc.).
 4. The method accordingto claim 2, wherein the liquid droplets are generated usingmicrofluidics technology and carry ingredients causing the liquiddroplets to form the inner phase and outer phases.
 5. The methodaccording to claim 3, wherein the hardened shell provides semi-permeablebarrier allowing for exchange of smaller molecular weight (MW) compounds(approximately 200,000 MW, and smaller) through the shell, but retentionof larger molecular weight compounds (approximately 300,000 MW, andlarger).
 6. The method according to claim 4, wherein the liquid dropletsare contained within a carrier oil.
 7. The method according to claim 4,wherein the liquid droplets contain an inner aqueous phase enriched inType 1 polymer and an outer aqueous phase enriched in Type II polymer.8. The method according to claim 7, wherein the Type I polymer isDextran and Type II polymer is modified polyethylene glycol polymer thatcan be cross-linked.
 9. The method according to claim 7, wherein thehigh concentricity of an aqueous two-phase system is achieved byreducing the density mismatch between two aqueous phases.
 10. The methodaccording to claim 1, wherein the compartment (liquid droplet, aqueoustwo-phase system droplet or capsule) is in the range from 1 μm to 100 μmsize, preferably in the range 10-60 μm and more preferably in the rangeof 20-40 μm.
 11. The method according to claim 1, wherein thebiochemical components, proteins, nuclei acids, bacteria, mammaliancells and other biological samples can be encapsulated within aplurality of liquid droplets, aqueous two-phase system droplets orcapsules.
 12. The method according to claim 11, wherein the encapsulatedspecies (e.g. cells, nucleic acids etc.) are treated with biochemicaland/or biological reagents.
 13. The method according to claim 11,wherein nucleic acid(s) or encapsulated cells is/are enzymaticallytreated.
 14. The method according to claim 11, wherein the capsulescarrying encapsulated molecules or cells can be processed throughmulti-step reactions and/or operations off-chip.
 15. The methodaccording to claim 11, wherein encapsulated species can be released fromthe compartment into surrounding fluid by breaking the compartment.